The advances seen with the use of anti-retroviral treatment, known as highly active anti-retroviral therapy (HAART), against HIV-1 are limited today by the cost and toxicity of lifelong administration of the drugs. An innovative therapeutic strategy has been proposed to boost the immune system of infected patients with HIV-1 vaccines and to help limit the use of HAART. However, the concept of therapeutic vaccination implies that the host immune system is still competent for eliciting an immune response after vaccination. Patients suffering from HIV-1 infection usually exhibit impaired immune defenses caused by the loss of the CD4+ T cells that are essential for mounting both the cell-mediated and antibody (Ab)-mediated immune responses. To overcome this challenge for current immunotherapeutic HIV-1 vaccine development, we propose to develop a heterologous prime-boost vaccine approach with the addition of CD40 ligand, a member of the tumor necrosis factor superfamily (TNFSF) of co-stimulatory molecules, in both the prime and boost phases of vaccination to increase antiviral responses in an immunocompromised host with deficiency of CD4+ T cell help. Recent studies in murine systems have demonstrated that CD4+ T cell help is required for antiviral CD8+ cytotoxic T lymphocyte (CTL) response through interactions with dendritic cells (DCs). Furthermore, the help from CD4+ T cells can be replaced or bypassed by ligation of CD40L with CD40 on DCs. We, therefore, hypothesize that employing CD40L as a molecular adjuvant in combination with vaccines may represent a novel strategy to induce potent antiviral CTL and Ab responses in CD4+ T cell-deficient states such as HIV-1 infection. We have two specific aims: 1) determine whether a heterologous DNA prime-ALVAC boost vaccine approach with the addition of CD40L as a molecular adjuvant in both the prime and boost phases of vaccination can improve HIV-1-specific CTL and Ab responses to vCP1452 (a currently licensed ALVAC-HIV vaccine) immunization in the absence of CD4+ T cell help in a CD4+ T cell-depleted murine model; 2) determine whether the DNA prime-ALVAC boost strategy with the addition of CD40L as a molecular adjuvant in both the prime and boost phases of vaccination can also improve the HIV vaccine vCP1452 immunogenicity in aged mice, whose CD4+ T cell function has been found impaired. This proposal is innovative in using molecular adjuvants to bypass CD4+ T cell help for the development of therapeutic HIV-1 vaccines for immunocompromised patients. The long-term goal of this study is to develop a novel therapeutic formulation of HIV-1 vaccines in combination with molecular adjuvants to induce more potent and long-lasting CTL and Ab responses in immunocompromised HIV-1-infected hosts. Plain language: This study will develop and evaluate a novel formulation of therapeutic HIV vaccine regimen in the absence of CD4+ T help in a mouse model, which roughly mimics an accelerated form of AIDS in humans. Data obtained from this study will provide key information for therapeutic vaccine development for young and aged people living with HIV/AIDS. [unreadable] [unreadable] [unreadable]